PROJECT SUMMARY Nearly 1 in 10 adults over the age of 65 in the U.S. suffer from atrial fibrillation (AF) leading to approximately $6 billion annually in healthcare costs. Because advanced age is a primary risk factor for developing AF, the overall incidence is expected to rise steadily over the coming decades as our population ages. Current therapeutic interventions have remarkably poor efficacy and/or untoward side effects due in large part to our inability to target the root cause of the disease and provide specificity for the atria and the patient. The central objective of this proposal is to create and validate a robust 3D microphysiological model of abnormal human atrial conduction using induced pluripotent stem cells from the patient. The model will simulate important elements of AF, such as conduction velocity, and develop novel therapeutic strategies that employ adenoviral delivery of gene interference (CRISPRi) that target altered gene regulatory pathways as the source of AF. We will accomplish this objective by completing the following specific aims: 1) characterize the transcriptome, epigenome, and electrophysiology of adult human atrial cardiomyocytes (normal and AF); 2) create a 3D in vitro disease model of human atrial conduction leveraging human iPS cell-derived atrial cardiomyocytes (iPS- aCM) and atrial regulatory gene expression; 3) design and test an adenoviral gene delivery strategy to specifically target atrial (not ventricular or nodal) cardiomyocytes; 4) demonstrate atrial specific adenoviral delivery (SA3) of CRISPRi and gene interference of PITX2 in ex vivo human atrial tissue; and 5) create iPS- aCM and the corresponding in vitro model of atrial conduction from a cohort of normal subjects and patients with AF; characterize drug efficacy and gene delivery in the in vitro models using a panel of existing drugs and our atrial specific adenoviral construct for CRISPR gene interference of PITX2 (SA3). Completing the specific aims will provide a model of human atrial conduction that can be used as a broad platform to understand drug efficacy and safety for diseases such as AF.!